Abstract During the past three and one-half years of R01 grant support, we have made substantial progress in: a) assessing the role of C3 in the newly discovered intracellular complement system (ICS or ?complosome?), including its modulation by CD46 signaling, b) developing an assay to quantitate a biologically active form of C3 known as C3(H2O), c) identifying a receptor-mediated C3(H2O) uptake process common to most cell types, and d) determining the ICS response in cells and an organ system (the airway) to ischemic and immune- mediated attack. Our immediate and ongoing goals are to publish our results describing the human receptor for C3(H2O) as well as studies demonstrating that C3 expression by lung transplants is reduced in alloimmune- mediated injury. We are also further delineating complosome activation and regulatory mechanisms at the RNA and protein level and are extending our analyses to human peripheral blood phagocytic, epithelial and, especially, endothelial cell populations. A further goal is to dissect the role of the ICS, utilizing both an in vitro model system with primary human cells isolated from lung tissue donors and corresponding human cell lines, as well as in mouse models of infection. Thus, we propose to continue to develop and employ model systems to obtain a detailed map of how a cell type's complosome functions in normal homeostasis as well as its response to cellular damage and malignancy. Such analyses will likely trigger new insights into how dysfunctions of this system correlate with human diseases. An important goal of this proposal relates to oncology. In addition to its role as an inhibitor of complement activation on host cells and a critical component of the complosome, CD46 is emerging as a key player in both malignant transformation and cancer immunotherapy. On one hand, CD46 is overexpressed on many tumors, yet on the other hand, it is targeted by therapeutic oncolytic viruses that use it as a docking mechanism. We have contributed to these studies and now plan to dissect the rationale of its overexpression and its effect on the complosome, beginning with multiple myeloma in collaboration with the Siteman Cancer Center. The models developed will be extended subsequently to other types of cancers. In summary, our proposal draws on the strength of our long-term commitment to the field of complement research and our ambitious undertakings related to defining the mechanisms and players of the newly discovered ICS. We are especially attracted to this grant mechanism because of its flexibility. This proposal draws not only on the expertise of the PI, but also on a team of talented individuals who are well positioned to continue being leaders the field by creating more innovative model systems and obtaining a comprehensive map that expands our knowledge-base relative to the ?workings? of the ICS.